Biogenic hemin-based mri contrast agents, and compositions and methods thereof

ABSTRACT

The invention provides a novel class of MRI contrast agents based on biogenic hemin and compositions and methods of preparation and use thereof.

PRIORITY CLAIMS AND RELATED APPLICATIONS

This application is a divisional of and claims the benefit of priorityto U.S. Ser. No. 16/608,838, filed Oct. 27, 2019, which is the U.S.national phase of and claims priority to PCT/US18/33074, filed May 17,2018, which claims the benefit of priority from U.S. ProvisionalApplication Ser. No. 62/509,208, filed on May 22, 2017, the entirecontent of each of which is incorporated herein by reference.

TECHNICAL FIELDS OF THE INVENTION

The invention generally relates to diagnostics and MRI contrast agents.More particularly, the invention relates to a novel class of MRIcontrast agents based on biogenic hemin.

BACKGROUND OF THE INVENTION

Magnetic resonance imaging (MRI), a medical imaging technique used inradiology, is one of the most used non-invasive, versatile imagingmodalities for clinical detection, staging, and monitoring of thetreatment of tumors. MRI affords unique advantages, including highspatial resolution, outstanding soft tissue contrast, and causes noradiation damage. Recently, the development of magnetic contrast agents(CAs) has improved the inherent sensitivity of MRI, enabling thistechnique to visualize specific biological processes at both cellularand molecular levels. (Sun, et al. 2008 Adv. Drug Delivery Rev. 60,(11), 1252-65; Zhang, et al. 2016 Nanoscale 8, (20), 10491-510.)

MRI, however, often encounters an inherent low sensitivity since thereis little difference between normal and abnormal soft tissues inrelaxation time and resulting contrast. To overcome this drawback,materials that possess magnetic properties, namely MRI contrast agents(MRI-CAs), have been used to enhance image quality and signal contrast.It is reported that more than 40% of all MRI examinations utilize acontrast agent. The development of biocompatible and effective MRI-CAsplays an important role in the application of MRI in clinical radiology.

Different magnetic agents have continuously emerged as contrast mediafor MRI, which are generally classified into two classes. One class issuperparamagnetic agents that shorten the T2 relaxation time and producenegative enhancement effects, such as Fe-based CAs such assuperparamagnetic iron oxide (SPIO) and ultrasmall superparamagneticiron oxide (USPIO). The other class is paramagnetic complexes thataccelerate the Ti relaxation process and possess positivelysignal-enhancing ability. Compared with the former, the latter CAs ownthe merits of signal-brighten effect, concomitant superiorsignal-to-noise ratio and no susceptibility artefact, thus becoming themost frequently used MRI-CAs in the clinic.

Existing T1 MRI-CAs, however, contain extrinsic metal elements such asgadolinium (Gd) and manganese (Mn), which raise certain biosafetyissues. For example, the clinically available Gd-based CAs suffer theshortcomings of short life spans in the body and potential toxicity offree Gd ions that are released from the complex. Meanwhile, intravenousadministration of gadolinium-based chelates is reported to inducegadolinium retention in tissues such as the brain, kidney and skin, andpotentially lead to serious complications, for example, nephrogenicsystemic fibrosis and organ functional failure. Mn-based CAs areextremely toxic to the liver and heart once free Mn ions are released,due to which Mn-based CAs are hardly used for clinical purposes.(Caravan, et al. 1999 Chem Rev. 99, (9), 2293-352; Na, et al. 2009 J.Mater. Chem. 19, (35), 6267; McDonald, et al. 2015 J. Radiology 275,(3), 772-82; Tu, et al. 2012 Wiley Interdiscip. Rev: Nanomed.Nanobiotechnol. 4, (4), 448-57.)

Thus, there is an ongoing need for novel MRI CAs that possess high T1relaxivity and excellent biosafety.

SUMMARY OF THE INVENTION

The invention provides a novel class of biogenic, nontoxic and effectiveT1 MRI contrast agents suitable for a wide range of clinicalapplications. Endogenous hemins derived from blood and conjugatesthereof are employed to serve as biogenic and gadolinium-free contrastagents for MR imaging.

The disclosed hemin-based contrast agents can be readily prepared byhydrating hemin in alkaline environment or conjugating hemin withhydrophilic ligands, leading to water-soluble and paramagnetic compoundswith remarkable enhancement of T1 effects in in vitro and in vivo MRI.In addition, the hemin-based MRI contrast agents are metabolized andcleared by the living body. The paramagnetic property, favorabletoxicity profile and biodegradable ability make this new class ofcontrast agents promising alternatives to Gd-based CAs in clinicalradiology.

In one aspect, the invention generally relates to a method for magneticresonance imaging (MRI). The method includes administering to a subjectin need thereof an aqueous composition comprising Fe³⁺-containing hemin.

In another aspect, the invention generally relates to an aqueouscomposition suitable for use as an MRI contrast agent. The aqueouscomposition is comprised of Fe³⁺-containing hemin.

In yet another aspect, the invention generally relates to a contrastagent for MRI that is comprised of Fe³⁺-containing hemin covalentlycoupled to an oligomer or polymer.

In yet another aspect, the invention generally relates to a method formagnetic resonance imaging. The method includes administering to asubject in need thereof an aqueous composition comprising aFe³⁺-containing hemin covalently coupled to an oligomer or polymer.

In yet another aspect, the invention generally relates to a method forpreparing a polymer conjugated and Fe³⁺-containing hemin. The methodincludes reacting hematin with an oligo(ethylene oxide) or poly(ethyleneoxide) modified with an amino group in the presence of a coupling agentin an aqueous solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph showing r1 values and 1/T1 values according to Feconcentrations of metalloporphyrin-based MRI-CAs after dispersion inwater.

FIG. 2 shows 3D images of MR angiography acquired after intravenousinjection to a mouse with metalloporphyrin-based MRI-CAs.

FIG. 3 shows body weight changes of mice according to time after tailvein injection of metalloporphyrin-based MRI-CAs or equal volumes ofphosphate-buffered saline (PBS).

FIG. 4 shows exemplary ¹H NMR spectra of hydrophilic HeOH-PEG.

FIG. 5 shows r1 values and 1/T1 values according to Fe concentrations ofHe—OH after dispersion in water.

FIG. 6 shows r1 values and 1/T1 values according to Fe concentrations ofHeOH-PEG after dispersion in DI water.

FIG. 7 shows T1-weighted MR images of HeOH-PEG particles according tovarious Fe concentrations.

FIG. 8 shows cell toxicities of HeOH-PEG to hela cell.

FIG. 9 shows body weight changes of mice according to time after tailvein injection of HeOH-PEG or equal volumes of PBS.

FIG. 10 shows hematoxylin and eosin stained tissue sections of the mainvisceral organs in mice at 14 day post-injection ofmetalloporphyrin-based MRI-CAs.

FIG. 11 shows a 3D image of MR angiography acquired from a mouse afterintravenous injection with HeOH-PEG.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based in part on the unexpected discovery of a novelclass of contrast agents based on endogenous and biogenic hemin that aresuitable for used in MRI.

MRI contrast agents play an important role in clinic examinations.Gd-based chelates are frequently used. Gd-based CAs suffer from severalkey shortcomings, including short life spans, insufficient targetingability, potential toxicity resulting from the release of free Gd ions,and high risk of Gd retention in the kidney, brain and neural tissues.It has long been desired to develop biogenic, nontoxic and efficientT1-MRI contrast agents for clinical applications.

The present invention exploits endogenous hemins derived from blood toafford a novel class of biogenic and gadolinium-free contrast agents forMR imaging. The present invention has demonstrated the paramagneticproperty and in vivo biosafety of the hemin-based CAs disclosed herein.For example, a biogenic hemin is shown herein to possesssignal-enhancing capability in T1-weighted MRI and is suitable to serveas a contrast agent for MR imaging.

The hemin-based CAs may be prepared, for example, by hydrating hemin inan alkaline environment or by conjugating hemin with hydrophilicligands. The resulting CAs are water-soluble and paramagnetic and showremarkable enhancement effects in in vitro and in vivo T1 MRI. Inaddition, the hemin-based MRI contrast agents of the invention arecleared by the living body. Taken together, the superior paramagneticproperty, biodegradable ability and favorable toxicity profile make thisnovel class of CAs a promising alternative to the traditional Gd-basedCAs in clinical diagnostic radiology.

Methemoglobin, which is composed of four iron-containing heme groupssurrounding a globin group, affect T1 relaxation and display the signalof significant brightness in T1-weighted MRI. This phenomenon isassociated with the special space configuration of methemoglobin, inwhich the iron in the heme group is in the Fe³⁺ (ferric) state, not theFe²⁺ (ferrous) state of normal hemoglobin.

This form of heme groups containing Fe³⁺ ions, called hemin, isrepresented aschloro[7,12-diethenyl-3,8,13,17-tetramethyl-21H,23H-porphine-2,18-dipropanoato(2-)-N,N,N,N] iron.

Hemes are most commonly recognized as components of hemoglobin, the redpigment in blood. Chemically, heme is a cofactor consisting of a Fe²⁺(ferrous) ion contained in the centre of a heterocyclic macrocycleorganic compound called porphyrin, which is made up of four pyrrolegroups joined together by methine bridges. Not all porphyrins containiron, but a substantial fraction of porphyrin-containing metalloproteinshas heme as the prosthetic group and are known as hemoproteins.

The structural formula for hemin is:

One of the most important biological functions of hemoproteins iselectron transfer, where the heme iron and the porphyrin molecule serveas an electron source in redox chemistry and peroxidase reactions,respectively. This ability of electron transfer makes it easy for theFe²⁺ ion of heme to lose their outer electrons and convert into Fe³⁺ ionwith five 3d electrons. The unpaired electrons confer the heme iron withits paramagnetic capacity to shorten T1 relaxation.

Hemin for injection, also known as Panhematin®, is an FDA-approved drugused in the management of porphyria attacks, particularly in acuteintermittent porphyria. The term “hematin” has been used to describe thechemical reaction product of hemin and sodium carbonate solution.Hematin is eliminated from the human body by the enterohepatic pathwayand the urinary system in the forms of bilirubin metabolites.

Heme, however, exhibits limited dispersibility in aqueous media atphysiological pH conditions, which has led to serious challenges in thedevelopment of hemin-based contrast-enhancing agents.

In addition, to utilize hemin-based CAs in biomedical imaging, twoadditional issues are important.

First, hemin is an enzyme inhibitor that limits the synthesis ofδ-aminolevulinic acid, which subsequently slow down the rate of thehepatic and/or marrow synthesis of porphyrin. The biosafety of hematinwas one of the key factors that restricted its application in vivo,especially for diagnostic purposes. As an enzyme inhibitor, overdose ofhemin could inhibit the hepatic and/or marrow synthesis of porphyrin,which may lead to anemia resulting from the limited production of redblood cells. An intravenous infusion of Panhematin® containing a dose of1 to 4 mg/kg/day of hematin was demonstrated to be safe for clinicapplications. An excessive hematin dose (e.g., 12.2 mg/kg) was reportedto induce reversible renal shutdown only in a rare case.

Second, further improvement of hematin's T1 relaxivity is desirable,especially for in vivo applications.

The present invention exploits PEGylation of hemin. The results showedthat PEGylated hemin possess excellent water solubility and T1relaxivity. In regard to the aqueous dispersibility, the PEGylated heminof the invention can be directly dispersed in alkaline solutions atabout pH 11. Importantly, when the pH of the alkaline solutioncontaining hemin was reduced back to 7, no changes occurred in thedispersibility of the soluble hemin. In addition, PEGylated hemin alsodecreased hemin uptake and its biological metabolism in the liver.

Moreover, more hemin molecules can be conjugated within one template,which may enhance their paramagnetic capacity and consequently reducethe required dose. Finally, the size of hemin-containing agents may beregulated below about 6 nm by controlling the PEGylation and the numberof the linked heme molecules in order to make the CAs metabolize via theurinary system while providing a sufficient circulation time for MRimaging.

Thus, in one aspect, the invention generally relates to a method formagnetic resonance imaging. The method includes administering to asubject in need thereof an aqueous composition comprisingFe³⁺-containing hemin.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

In certain embodiments, the aqueous composition comprisingFe³⁺-containing hemin is formed, prior to administration, by dispersinghemin in an aqueous solution having a pH in the range from about 5 toabout 14 (e.g., from about 5 to about 14, from about 6 to about 14, fromabout 7 to about 14, from about 8 to about 14, from about 9 to about 14,from about 10 to about 14, from about 11 to about 14, from about 12 toabout 14, from about 13 to about 14, from about 5 to about 13, fromabout 5 to about 12, from about 5 to about 11, from about 5 to about 10,from about 5 to about 9, from about 5 to about 8, from about 5 to about7, from about 8 to about 12, from about 9 to about 13).

The unit dosage for administration may be adjusted depending on theactual MRI application. In certain embodiments, the unit dosage is fromabout 0.1 mg/kg to about 1 g/kg (e.g., from about 0.1 mg/kg to about 0.5g/kg, from about 0.1 mg/kg to about 0.1 g/kg, from about 0.1 mg/kg toabout 50 mg/kg, from about 0.1 mg/kg to about 20 mg/kg, from about 0.1mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 5 mg/kg, fromabout 0.1 mg/kg to about 1 mg/kg, from about 1 mg/kg to about 1 g/kg,from about 5 mg/kg to about 1 g/kg, from about 10 mg/kg to about 1 g/kg,from about 50 mg/kg to about 1 g/kg, from about 0.1 g/kg to about 1g/kg, from about 0.5 g/kg to about 1 g/kg, from about 200 mg/kg to about800 mg/kg, from about 400 mg/kg to about 800 mg/kg).

The method of MRI may be used to image any suitable organ or tissue ofthe body of a mammal (e.g., human). Exemplary organs and tissues thatmay be imaged include the heart, liver, spleen, lung, kidney, brain,eyes, colon, prostate and stomach.

In another aspect, the invention generally relates to an aqueouscomposition suitable for use as MRI contrast agent. The aqueouscomposition is comprised of Fe³⁺-containing hemin.

In yet another aspect, the invention generally relates to a contrastagent for MRI. The contrast agent is a Fe³⁺-containing hemin covalentlycoupled to an oligomer or polymer.

In certain embodiments of the contrast agent, the oligomer or polymer isan oligo(ethylene oxide) or poly(ethylene oxide). In certain embodimentsof the contrast agent, the contrast agent is formed by reacting hematinwith an oligo(ethylene oxide) or poly(ethylene oxide) modified with anamino group in the presence of a coupling agent.

In certain embodiments of the contrast agent, the oligo(ethylene oxide)or poly(ethylene oxide) has a molecular weight in the range from about80 to about 8,000,000 (e.g., from about 80 to about 5,000,000, fromabout 80 to about 2,000,000, from about 80 to about 1,000,000, fromabout 80 to about 500,000, from about 80 to about 200,000, from about 80to about 100,000, from about 80 to about 50,000, from about 80 to about10,000, from about 80 to about 5,000, from about 500 to about 8,000,000,from about 1,000 to about 8,000,000, from about 5,000 to about8,000,000, from about 10,000 to about 8,000,000, from about 50,000 toabout 8,000,000, from about 100,000 to about 8,000,000, from about500,000 to about 8,000,000, from about 1,000,000 to about 8,000,000,from about 1,000 to about 20,000, from about 20,000 to about 500,000,from about 50,000 to about 200,000).

In certain embodiments of the contrast agent, the oligomer or polymer isselected from chitosan, dendrimers, dextrin, peptides,poly(vinylpyrrolidone-co-dimethyl maleic acid), polysaccharide,polyacrylic acid, hyaluronic acid, liposomes, or protein (e.g., selectedfrom human serum albumin, bovine serum albumin, lysozyme,immunoglobulin, ferritin, antibodies and transferrin).

In yet another aspect, the invention generally relates to a method formagnetic resonance imaging. The method includes administering to asubject in need thereof an aqueous composition comprising aFe³⁺-containing hemin covalently coupled to an oligomer or polymer.

In certain embodiments of the method, the oligomer or polymer is anoligo(ethylene oxide) or poly(ethylene oxide). In certain embodiments ofthe method, the contrast agent is formed by reacting hematin with anoligo(ethylene oxide) or poly(ethylene oxide) modified with an aminogroup in the presence of a coupling agent.

In certain embodiments of the method, the oligo(ethylene oxide) orpoly(ethylene oxide) has a molecular weight in the range from about 80to about 8,000,000 (e.g., from about 80 to about 5,000,000, from about80 to about 2,000,000, from about 80 to about 1,000,000, from about 80to about 500,000, from about 80 to about 200,000, from about 80 to about100,000, from about 80 to about 50,000, from about 80 to about 10,000,from about 80 to about 5,000, from about 500 to about 8,000,000, fromabout 1,000 to about 8,000,000, from about 5,000 to about 8,000,000,from about 10,000 to about 8,000,000, from about 50,000 to about8,000,000, from about 100,000 to about 8,000,000, from about 500,000 toabout 8,000,000, from about 1,000,000 to about 8,000,000, from about1,000 to about 20,000, from about 20,000 to about 500,000, from about50,000 to about 200,000).

In certain embodiments of the method, the oligomer or polymer isselected from chitosan, dendrimers, dextrin, peptides,poly(vinylpyrrolidone-co-dimethyl maleic acid), polysaccharide,polyacrylic acid, hyaluronic acid, liposomes or protein (e.g., selectedfrom human serum albumin, bovine serum albumin, lysozyme,immunoglobulin, ferritin, antibodies, transferrin).

The unit dosage is dependent on the actual MRI application. In certainembodiments, the unit dosage for administration is from about 0.1 mg/kgto about 20 g/kg (e.g., 0.1 mg/kg to about 10 g/kg, 0.1 mg/kg to about 1g/kg, 0.1 mg/kg to about 500 mg/kg, 0.1 mg/kg to about 100 mg/kg, 0.1mg/kg to about 20 mg/kg, 0.1 mg/kg to about 5 mg/kg, 0.1 mg/kg to about1 mg/kg, 1 mg/kg to about 20 g/kg, 10 mg/kg to about 20 g/kg, 100 mg/kgto about 20 g/kg, 500 mg/kg to about 20 g/kg, 1 g/kg to about 20 g/kg, 5g/kg to about 20 g/kg, 10 mg/kg to about 1 g/kg, 100 mg/kg to about 500mg/kg).

The method of MRI may be applied to image any suitable organ or tissue,for example, selected from heart, liver, spleen, lung, kidney, brain,eyes, colon, prostate and stomach.

In yet another aspect, the invention generally relates to a method forpreparing a polymer conjugated, Fe³⁺-containing hemin. The methodincludes reacting hematin with an oligo(ethylene oxide) or poly(ethyleneoxide) modified with an amino group in the presence of a coupling agentin an aqueous solution.

In certain embodiments, the coupling reaction is conducted in thepresence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-hydroxysuccinimide (NHS). In certain embodiments, the oligo(ethyleneoxide) or poly(ethylene oxide) has a molecular weight in the range fromabout 80 to about 8,000,000 (e.g., from about 80 to about 5,000,000,from about 80 to about 2,000,000, from about 80 to about 1,000,000, fromabout 80 to about 500,000, from about 80 to about 200,000, from about 80to about 100,000, from about 80 to about 50,000, from about 80 to about10,000, from about 80 to about 5,000, from about 500 to about 8,000,000,from about 1,000 to about 8,000,000, from about 5,000 to about8,000,000, from about 10,000 to about 8,000,000, from about 50,000 toabout 8,000,000, from about 100,000 to about 8,000,000, from about500,000 to about 8,000,000, from about 1,000,000 to about 8,000,000,from about 1,000 to about 20,000, from about 20,000 to about 500,000,from about 50,000 to about 200,000).

The following examples are meant to be illustrative of the practice ofthe invention and not limiting in any way.

EXAMPLES

Hematin was studied for its properties in MR imaging. T1-weighted invitro imaging was carried out on a 3.0 T MR scanner, demonstrating theconcentration-dependent brightening effect of the prepared hematin.

FIG. 1 is a graph showing r1 values and 1/T1 values according to Feconcentrations of metalloporphyrin-based MRI-CAs after dispersion inwater. As shown in FIG. 1 , the T1 relaxivity value of hematin wasmeasured to be 0.424 mM⁻¹s⁻¹. To further evaluate its MRI performance invivo, hematin at a dose of 2 mg/kg/day, which is within the recommendedrange for clinical use, was administered into the tail veins of nudemice.

Following intravenous administration, an increase in the T1 signal wasclearly observed in the heart and large vessel (FIG. 2 ), demonstratingthe excellent signal enhancement of hematin for MRI. FIG. 2 shows the 3Dimages of MR angiography acquired from the mouse after intravenousinjection with metalloporphyrin-based MRI-CAs.

The biocompatibility of hematin for injection was comprehensivelyinvestigated, for example, via blood routine examination, histologicalanalysis, and body weight monitoring.

Table 1 shows the results of blood routine examination acquired frommice intravenous-injected with (Hemin+sodium bicarbonate). All theparameters of blood routine examination at different time pointspost-injection were within the normal range. Another group of miceinjected with PBS were used as control. As shown, almost all theparameters such as WBC, RBC, HGB, PLT, HCT, MCV, MCH, and MCHC, arewithin the normal range.

TABLE 1 Blood routine examination acquired from miceintravenous-injected with (Hemin + sodium bicarbonate) Normal RangeControl 6 h 12 h 24 h 3 d 5 d 10 d 14 d WBC (K/μL)  1.8-10.7 2.14 4.564.76 2.44 6.86 3.84 3.92 4.08 NE (K/μL) 0.1-2.4 0.60 0.85 0.71 0.28 0.790.87 0.96 0.43 LY(K/μL) 0.9-9.3 1.48 2.95 3.37 1.89 5.35 2.39 2.54 3.23MO (K/μL) 0.0-0.4 0.04 0.37 0.28 0.22 0.39 0.34 0.35 0.32 EO (K/μL)0.0-0.2 0.01 0.05 0.07 0.04 0.09 0.17 0.04 0.07 BA (K/μL) 0.0-0.2 0.010.03 0.02 0.01 0.03 0.08 0.03 0.03 RBC (M/μL) 6.36-9.42 9.22 7.29 7.826.31 7.73 7.20 8.72 7.14 HGB (g/dL) 11.0-15.1 13.9 11.2 12.3 11.0 11.413.1 12.4 10.5 HCT (%) 35.1-45.4 46.6 35.9 40.7 43.6 37.7 41.2 45.0 44.8MCV (fL) 45.4-60.3 50.5 49.2 52.0 50.6 48.2 54.0 51.6 48.8 MCH (pg)14.1-19.3 15.1 15.4 15.7 16.2 14.6 18.1 14.2 14.7 MCHC (K/μL) 30.2-34.229.8 31.2 30.2 32.1 30.2 31.2 30.6 30.2 RDW (K/μL) 12.4-27.0 17.5 17.216.0 15.3 17.9 21.7 17.4 18.4 PLT (K/μL)  592-2972 749 643 693 750 963826 967 617 MPV (fL)  5.0-20.0 5.4 5.9 6.0 6.1 5.9 6.5 5.6 5.4 Whiteblood cell (WBC), Neutrophils (NE), Lymphocytes (LY), Monocytes (MO),Eosinophils (EO), Basophils (BA), Red blood cell (RBC), Hemoglobin(HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscularhemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Redblood cell distribution width (RDW), Platelet Thrombocyte (PLT), Meanplatelet volume (MPV).

FIG. 3 shows body weight changes of mice according to time after tailvein injection of metalloporphyrin-based MRI-CAs or equal volumes ofPBS. The histopathological results showed no obvious histologicalchanges in several susceptible organs (e.g., heart, liver, spleen, lung,and kidney) for 14 days after administration. Moreover, the body weightexhibited no significant changes compared to the control group (FIG. 3). The results taken together demonstrate the excellent biocompatibilityof hematin for injection in vivo.

Hematin With PEGylation as MRI CAs

A MRI contrast agent was formed by conjugation of hematin with anamphiphilic material (in this case PEG2000) in an organic solvent usingcoupling agents (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-hydroxysuccinimide (NHS)). The formed hydrophilic He-PEG moleculeswere stable.

In a typical synthesis, 5 mg hematin was dispersed in 2 mLtetrahydrofuran (THF). EDC (5 mg) and NHS (5 mg) were added to thesolution to activate the carboxyl groups of hematin. After stirringvigorously for 10 min, 12 mg PEG2000 modified with amino group on thesurface, was added to the above mixture. The reaction mixture was keptat 4° C. in the dark for 12 h with continuous stirring. Redprecipitation was observed, which indicated the formation of He-PEGcrystals. Following the reaction, 2 mL distilled water was poured intothe organic solution to make all the precipitation of He-PEG crystalsdissolve.

Then, the organic solvent was removed in a rotary evaporator at reducedpressure at 4° C. for 4.0 h. Finally, the resulting He-PEG was purifiedby centrifugal ultrafiltration with a centrifuge filter tube (AmiconUltra-15 Centrifugal Filter Unit with Ultracel-30 membrane, 30 KDa,Millipore) at 5,500 rpm for 60 min. Large nanoparticles precipitated andthe supernatant containing small He-PEG was decanted, re-dissolved with2 mL of PBS buffer (pH 7.4, 10 mM) and stored at 4° C. for the followingexperiments.

The composition of the prepared He-PEG was confirmed by NMRspectroscopy. FIG. 4 shows a ¹H NMR spectrum of hydrophilic HeOH-PEGaccording to an embodiment of the present invention. The ¹H NMR spectrumof the He-PEG nanoparticles confirmed the successful conjugation of Hewith PEG2000. The magnetic property of the produced He-PEG particles wasdetermined by measuring the longitudinal relaxivity values, whichreferred to the slope of the 1/T1 (R1) plot versus Fe ion concentration.

FIGS. 5 and 6 show r1 values and 1/T1 values according to Feconcentrations of He—OH after dispersion in water. FIG. 7 showsT1-weighted MR images of HeOH-PEG particles according to various Feconcentrations. The r1 value of the He-PEG particles was found to be2.36 s⁻¹ mM⁻¹ Fe (FIG. 5 ), which was significantly higher than that ofHe—OH (0.579 s⁻¹ mM⁻¹ Fe, FIG. 6 ) and He—CO₃ (0.424 s⁻¹ mM⁻¹ Fe, FIG. 1). Meanwhile, T1-weighted MR images of He-PEG particles with various Feconcentrations are shown in FIG. 7 .

HeOH-PEG exhibited strong MR signals at low Fe concentrations. The highr1 values and excellent MR imaging capability showed that the preparedHe-PEG particles can serve as a positive MR contrast agent. Importantly,HeOH-PEG exhibited outstanding stability since no visible aggregation orsignificant change in the longitudinal relaxivity was observed after3-month storages in deionized (DI) water or phosphate-buffered saline(PBS).

In vitro and in vivo toxicity profiles of HeOH-PEG were investigatedbefore application of HeOH-PEG in MR imaging of animal models. FIG. 8shows results of cell toxicity test of HeOH-PEG using HeLa cell. Thecytotoxicity of the HeOH-PEG was examined via the MTT assay(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Variousconcentrations of HeOH-PEG were used to incubate with HeLa cells. Cellviabilities of HeLa cells were not affected by HeOH-PEG when compared tothose treated with PBS (FIG. 8 ), demonstrating the negligible toxicityof HeOH-PEG in vitro.

Additionally, HeOH-PEG also exhibited good biocompatibility in vivo.FIG. 9 shows body weight changes of mice according to time after tailvein injection of HeOH-PEG or equal volumes of PBS. After injection ofHeOH-PEG, the mice showed no obvious body weight loss during anobservational period of 2 weeks.

Meanwhile, blood samples were collected at different intervals aftertreatment with HeOH-PEG. Table 2 shows the results of blood routineexamination acquired from mice intravenous-injected with HeOH-PEG.Another group of mice injected of PBS were used as control. As shown,almost all the parameters such as WBC, RBC, HGB, PLT, HCT, MCV, MCH, andMCHC, were within the normal range. These results confirmed that almostall hematological parameters were within the normal range (Table 2).

Subsequently, hematoxylin and eosin examinations were performed on majororgans (e.g., heart, liver, spleen, lung, and kidney) collected fromhealthy mice and those treated with HeOH-PEG. The structures of thestudied organs appeared normal and showed no tissue damage,inflammation, or necrosis. FIG. 10 shows samples of hematoxylin andeosin stained tissue sections of the main visceral organs in mice at 14days post-injection of metalloporphyrin-based MRI-CAs. No significantdifferences were found between the cellular morphology of the studiedorgans in the HeOH-PEG treated group and the control group (FIG. 10 ).

These results together demonstrated that HeOH-PEG processed negligibleimmunogenicity and toxicity.

TABLE 2 Blood routine examination acquired from miceintravenous-injected with HeOH-PEG Normal range Control 6 h 12 h 24 h 3d 5 d 10 d 14 d WBC (K/μL)  1.8-10.7 4.36 5.14 4.86 4.92 5.00 3.22 3.383.80 NE (K/μL) 0.1-2.4 0.50 1.27 1.61 1.65 2.14 0.22 1.07 0.26 LY (K/μL)0.9-9.3 3.68 3.02 2.58 3.07 2.77 2.78 1.79 2.99 MO (K/μL) 0.0-0.4 0.160.35 0.25 0.12 0.08 0.19 0.31 0.29 EO (K/μL) 0.0-0.2 0.01 0.08 0.03 0.060.01 0.01 0.17 0.09 BA (K/μL) 0.0-0.2 0.00 0.02 0.00 0.02 0.00 0.01 0.050.04 RBC (M/μL) 6.36-9.42 6.38 8.02 8.86 8.94 9.64 6.29 8.24 6.88 HGB(g/dL) 11.0-15.1 11.3 12.5 13.8 14.6 15.8 10.8 12.0 11.3 HCT (%)35.1-45.4 41.6 41.3 46.5 45.3 46.2 44.8 45.8 35.4 MCV (fL) 45.4-60.349.6 51.5 52.5 52.9 52.8 56.1 55.6 51.5 MCH (pg) 14.1-19.3 14.6 15.615.6 16.3 15.8 15.8 14.6 16.4 MCHC (K/μL) 30.2-34.2 29.4 30.3 29.7 30.929.9 28.2 30.2 31.9 RDW (K/μL) 12.4-27.0 17.0 16.5 16.1 16.7 17.1 22.218.2 18.6 PLT (K/μL)  592-2972 593 890 671 632 570 700 683 751 MPV (fL) 5.0-20.0 5.6 5.9 5.5 6.0 6.3 5.9 6.7 5.7

To study the potential application of HeOH-PEG for in vivo MRI, a seriesof MR imaging were performed at different time points after HeOH-PEG wasinjected via tail vein into health node mice (500 μL, 0.48 mmol Au perkg mice). FIG. 11 is the 3D image of MR angiography acquired from themouse after intravenous injection with HeOH-PEG.

As shown in FIG. 11 , MR signal in the great vessels of the mice becamestronger as early as 5 min post-injection by comparison with the initialMR images prior to injection. At 20 min post injection, the HeOH-PEGparticles flowed through the vessels in liver and kidney, making thekidney contour and the liver brighten. The MR signal enhancement withexcellent contrast in the vasculature lasted until 60 min postinjection. Then, the vascular MR signal gradually decreased and most ofthe HeOH-PEG particles were mainly accumulated in kidney, resulting inrenal MR signal enhancement. At 2 h post injection, the bladderexhibited remarkable hyperintensity, indicating that the HeOH-PEGparticles were excreted from the body by the urinary system. After 24 hpost injection, the probe was mostly cleared from the body as the MRsignal of the whole body returned back to the baseline level. The invivo MRI results showed that HeOH-PEG can indeed act as an efficient andclearable contrast agent for MR imaging.

Applicant's disclosure is described herein in preferred embodiments withreference to the Figures, in which like numbers represent the same orsimilar elements. Reference throughout this specification to “oneembodiment,” “an embodiment,” or similar language means that aparticular feature, structure, or characteristic described in connectionwith the embodiment is included in at least one embodiment of thepresent invention. Thus, appearances of the phrases “in one embodiment,”“in an embodiment,” and similar language throughout this specificationmay, but do not necessarily, all refer to the same embodiment.

The described features, structures, or characteristics of Applicant'sdisclosure may be combined in any suitable manner in one or moreembodiments. In the description, herein, numerous specific details arerecited to provide a thorough understanding of embodiments of theinvention. One skilled in the relevant art will recognize, however, thatApplicant's composition and/or method may be practiced without one ormore of the specific details, or with other methods, components,materials, and so forth. In other instances, well-known structures,materials, or operations are not shown or described in detail to avoidobscuring aspects of the disclosure.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference, unless the context clearlydictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although any methods and materials similar or equivalent tothose described herein can also be used in the practice or testing ofthe present disclosure, the preferred methods and materials are nowdescribed. Methods recited herein may be carried out in any order thatis logically possible, in addition to a particular order disclosed.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes. Any material, or portion thereof, that is said to beincorporated by reference herein, but which conflicts with existingdefinitions, statements, or other disclosure material explicitly setforth herein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.In the event of a conflict, the conflict is to be resolved in favor ofthe present disclosure as the preferred disclosure.

Equivalents

The representative examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the examples andthe references to the scientific and patent literature included herein.The examples contain important additional information, exemplificationand guidance that can be adapted to the practice of this invention inits various embodiments and equivalents thereof.

1. A method for magnetic resonance imaging (MRI), comprisingadministering to a subject in need thereof an aqueous compositioncomprising Fe³⁺-containing hemin.
 2. The method of claim 1, wherein theaqueous composition comprising Fe³⁺-containing hemin is formed prior toadministration by dispersing hemin in an aqueous solution having a pH inthe range from about 5 to about
 14. 3. The method of claim 1, whereinthe unit dosage of administration is from about 0.1 mg/kg to about 1g/kg.
 4. The method of claim 1, wherein the MRI is for an organ selectedfrom heart, liver, spleen, lung, kidney, brain, eyes, colon, prostateand stomach. 5-22. (canceled)